Two-photon fluorescence microscopy (Denk, Strickler, Webb, U.S. Pat. No. 5,034,613) and confocal-fluorescence microscopy (See "Confocal Microscopy" SPIE Milestone Series, 1996, ISBN 0-8194-2372-6, edited by Barry Master for original papers and patents) for three-dimensional optical imaging have made a significant impact in many disciplines, especially cell biology. Fluorescence microscopy requires either intrinsic fluorescence or staining with a fluorescent dye. Both of these methods lack generality, and adding dyes affects the properties of biological specimens. In confocal microscopy a pinhole in the optical path restricts detection to a small focal volume element, thereby efficiently reducing signal background and improving image contrast. In two-photon microscopy the excitation volume is restricted by virtue of a nonlinear process in which two photons from a pulsed laser source are simultaneously absorbed. Both of these techniques allow the reconstruction of three-dimensional images.
For chemical species or cellular components that either do not fluoresce or cannot tolerate labeling, Infrared (IR) microscopy or spontaneous Raman microscopy can be used. In these cases, vibrational spectroscopy provides the contrast mechanism. Direct imaging using IR light absorption in a microscope has been used. However, the spatial resolution of this technique is low (.about.10 .mu.m) due to the long wavelength of the light used. Three-dimensional spontaneous Raman microscopy of biological samples has been demonstrated with a confocal microscope as reported by Puppels, G. J., de Mul, F. F. M., Otto, C., Greve, J., Robert-Nicoud, M., Arndt-Jovin, D. J. & Jovin, T. M. Studying single living cells and chromosomes by confocal Raman microspectroscopy. Nature 347, 301-303 (1990); Sijtsema, N. M, Wouters, S. D., De Grauw, C. J., Otto, C. & Greve, J. Confocal direct imaging Raman microscope: Design and applications in biology. Appl. Spectrosc., 52, 348-355 (1998). The intrinsically weak Raman signal necessitates high laser powers (typically&gt;10 mW) and is often overwhelmed by the fluorescence background of the sample.
Two nonlinear techniques have been demonstrated for vibrational spectroscopy. The first technique is Coherent Anti-Stokes Raman Scattering (CARS), described e.g. in Shen, Y. R. The Principles of Nonlinear Optics (John Wiley & Sons Inc. New York, 1984),267-275, is a nonlinear optical four-wave-mixing process containing vibrational spectroscopic information. A schematic diagram of this process is given in FIG. 1A (prior art). For CARS spectroscopy, a pump laser and a Stokes laser beam, with center frequencies of .omega.p and .omega.s, respectively, are spatially overlapped. When the frequency difference v.sub.p -v.sub.s coincides with the frequency of a vibrational transition of the sample, a strong CARS signal at v.sub.as =2v.sub.p -v.sub.s is generated in a direction determined by the phase-matching conditions (FIG. 1A.) The intensity of the detected signal, I.sub.AS, is proportional to the square modulus of a molecular term .chi..sup.(3) times the pump intensity I.sub.P squared, times the Stokes wave intensity I.sub.S. EQU I.sub.AS =I.sub.P.sup.2 .multidot.I.sub.S .multidot..vertline..chi..sup.(3) .vertline..sup.2
.chi..sup.(3) includes terms for a resonance enhancement at certain molecular vibration frequencies. Because the process is cubic in laser power, the signal is only generated efficiently with high excitation intensities. It is therefore advantageous to use high peak power which are readily available from femtosecond or picosecond light pulses.
U.S. Pat. No. 4,405,237 to Manuccia and Reintjes discusses a Coherent Anti-Stokes Raman spectroscopy device for microscopic imaging or observing cellular constituents in live samples. In their proposed scheme, two parallel, but not overlapping laser beams are focused to a common focal spot. The two laser beams are provided by two lasers tunable to wavelengths of 565-620 nm and 620-700 nm respectively as reported in Scanning coherent anti-Stokes Raman microscope, M D Duncan, J Reintjes, T J Manuccia, Optics Letters, Vol. 7, No. 8, Aug. 1982. They collect and detect the signal beam in the phase matched condition. In this case, however, the cone angles of the two beams are small, which means that the size of the focal spot is large (10 micron Duncan et al., 1982), and the spatial resolution is low. This was not a confocal arrangement therefore high resolution three-dimensional sectioning capability was not achievable. Moreover, the sensitivity of CARS microscopy was limited by the nonresonant background signal. The magnitude of the non-resonant background signal is dependent on the wavelengths of the excitation lasers. With visible wavelength lasers, the CARS signal is dominated by the non-resonant background signal. These intrinsic difficulties have limited the experimental demonstration and application of this proposed scheme.
An article by Zhao et al., The wave-mixing near field optics amplifiers: a theoretical feasibility study for non-linear NFO experiments in biology chemistry and materials science, Elsevier, Ultramicroscopy 61 (1995) 69-80, discusses a method of using near field optics to conduct nonlinear spectroscopy, including Coherent Anti-Stokes Raman spectroscopy. Zhao et al. proposed wave mixing with a first laser light at a first frequency and a second laser light at a second frequency that both impinge on a sample slab. The feasibility was discussed for a situation in which the first laser light emerges from an aperture probe and the second laser light be incident at a non-normal angle. This configuration for near-field CARS, intending for a high spatial resolution beyond the diffraction limit, requires a feedback system regulating the probe-sample distance and has not yet been experimentally demonstrated.
The second nonlinear vibrational spectroscopic technique is Sum Frequency Generation (SFG), as described e.g. in Shen, Y. R. The Principles of Nonlinear Optics (John Wiley & Sons Inc. New York, 1984), 67--85. Sum Frequency Generation is a nonlinear process requiring two incident laser beams with frequency of v.sub.s and v.sub.p focused to a common spot, generating a new frequency v.sub.sf =v.sub.s +v.sub.p and providing vibrational contrast when v.sub.s is on resonance with molecular vibration. The energy diagram and phase matching diagram are shown in FIG. 1B. Distinctions of SFG from CARS include (1) The SFG process is a .chi..sup.(2) effect and depends linearly on the intensity of each incident beam, (2) The sensitivity of SFG to vibration requires one of the lasers be infrared (IR), so that its frequency (v.sub.1) is in resonance with that of a vibrational transition of the sample (3) the IR beam is absorbed by the sample (4) the signal beam is produced only at a surface, which is very useful for surface specific information generally unavailable by other means. Microscopy with SFG has not been demonstrated.
Imaging with third harmonic generation, another coherent four-wave-mixing process, has been demonstrated by Barad, Y., Eisenberg, H., Horowitz, M. & Silberberg, Y. Nonlinear scanning laser microscopy by third harmonic generation Appl. Phys. Lett. 70, 922-924 (1997); Muller, M., Squier, J., Wilson, K. R. & Brakenhoff G. J. 3D-microscopy of transparent objects using third-harmonic generation. J. Microsc., 191, 266 (1998) This technique is similar to CARS microscopy in that the signal is dependent on the third-order polarizability .chi..sup.(3), but differs in that it probes the electronic contributions to .chi..sup.(3) not specifically sensitive to vibrational properties. The electronic contributions to .chi..sup.(3) are exhibited as a weak non-resonant background signal in CARS microscopy.
Hence, there is a need in the art for a method and apparatus for nonlinear vibrational microscopy that is sensitive to vibrational properties, has high sensitivity and high spatial resolution, and has a straightforward implementation.